Raczynska_2026_Int.J.Biol.Macromol_357_151616

The enzymatic degradation of synthetic polymers offers a sustainable route for plastic waste recycling, yet progress remains limited by an incomplete understanding of enzyme-polymer recognition and catalytic mechanisms. While cutinases have shown potential to degrade polyester-based polyurethanes (PURs), the molecular determinants of substrate binding and selectivity are still poorly resolved. In this study, we combined computational and experimental approaches to elucidate the interaction between Thermobifida fusca cutinase (TfCut2) and Impranil DLN, a polyester-based PUR widely used as a model substrate. NMR analysis refined the structural description of Impranil DLN, enabling the construction of accurate molecular models for simulations. Using computational analyses, we identified multiple catalytically competent TfCut2-Impranil complexes and characterised the key residues stabilising the substrate in the active site, including Y60, M131, and W155 forming an "aromatic clamp" near the oxyanion hole. The small differences in binding free energy across ligand poses suggest flexible substrate accommodation rather than a single preferred orientation, consistent with the enzyme's broad specificity. Guided by computational insights, targeted mutations near the active site were re-designed and experimentally tested. Notably, variants T61V, G62A, and T207D exhibited enhanced Impranil DLN degradation rates, with G62A showing over a twofold increase in initial rate and T207D demonstrating improved thermal stability and production yield. These findings underscore that efficient PUR degradation arises not from strong binding alone but from the enzyme's ability to guide the polymer into productive orientations while preserving catalytic geometry and turnover. This mechanistic understanding offers clearer design principles for improving PUR hydrolases.